Ectopic Integration Vectors for Generating Fluorescent Promoter Fusions in <i>Bacillus subtilis</i> with Minimal Dark Noise

<div><p>Fluorescent protein promoter reporters are important tools that are widely used for diverse purposes in microbiology, systems biology and synthetic biology and considerable engineering efforts are still geared at improving the sensitivity of the reporter systems. Here we focus on dark noise, i.e. the signal that is generated by the empty vector control. We quantitatively characterize the dark noise of a few common bacterial reporter systems by single cell microscopy. All benchmarked reporter systems generated significant amounts of dark noise that exceed the cellular autofluorescence to different extents. We then reengineered a multicolor set of fluorescent ectopic integration vectors for <i>Bacillus subtilis</i> by introducing a terminator immediately upstream of the promoter insertion site, resulting in an up to 2.7-fold reduction of noise levels. The sensitivity and dynamic range of the new high-performance pXFP_Star reporter system is only limited by cellular autofluorescence. Moreover, based on studies of the <i>rapE</i> promoter of <i>B. subtilis</i> we show that the new pXFP_Star reporter system reliably reports on the weak activity of the <i>rapE</i> promoter whereas the original reporter system fails because of transcriptional interference. Since the pXFP_Star reporter system properly isolates the promoter from spurious transcripts, it is a particularly suitable tool for quantitative characterization of weak promoters in <i>B. subtilis</i>.</p></div

Strains and plasmids.

<p>* two independent clones are used for this study.</p

Map of the high-performance <i>amyE</i> integration vectors pXFP_Star.

<p>The pXFP_Star vector set features the transcriptional terminator of the <i>B. subtilis gyrA</i> gene and a small cloning site (SCS) upstream of the LIC site used for ligation-independent cloning of promoter inserts. The grey cross marks the position of the deleted <i>ldh</i> parts. The vector variants differ only with respect to the fluorophore <i>gfpmut3</i>, <i>iyfp</i> or <i>cfp_Bs</i>. The other elements, annotated as <i>amyE</i>′ =  <i>amyE</i> front, ′<i>amyE</i> =  <i>amyE</i> back, denote homologous <i>amyE</i> parts enabling chromosomal integration into the <i>amyE</i> locus. β<i>-lactamase</i> gene (<i>bla</i>) and <i>chloramphenicol acetyltransferase</i> gene (<i>cat</i>) are required for selection in <i>E. coli</i> and <i>B. subtilis</i> respectively. The grey arrow denotes the ColE1 region required for plasmid replication with the ColE1 origin of DNA replication (+1) highlighted in magenta.</p

Dark noise benchmarking characteristics.

<p>The benchmarking parameters D and S are defined as the ratio of the mean intensities (μ) and the standard deviations (σ) of the respective intensity distributions originating from the empty vector control (EV) and the autofluorescence (AF) of the parental strain. D is correlated to the accessible dynamic range and S is correlated to the sensitivity of the reporter system.</p

D and S characteristics for pXFP_Star family at OD_600nm of 5.

<p>D and S characteristics for pXFP_Star family at OD_600nm of 5.</p

Analysis of <i>rapE</i> promoter fusions with the pGFP_Star and pGFPamy reporter system.

<p>Histograms of the fluorescence intensity distribution for the <i>rapE</i> promoter fusion (P<i>rapE</i>) and the “empty” vector reference strain (EV) obtained with the pGFP_Star (top panel) and the pGFPamy (bottom panel) reporter system, respectively. The left column shows the results measured at OD_600nm = 2.5 when the <i>rapE</i> promoter is repressed and the right panel shows results obtained at OD_600nm = 6 when the promoter is weakly active. For the pGFPamy reporter system <i>PrapE</i> cells produce less fluorescence than the EV. Hence, spurious upstream transcription and <i>rapE</i> promoter transcription do not contribute additively to the fluorescence signal but interfere with each other in unpredictable manners. The Star-system is capable to reliably report on <i>rapE</i> promoter activity.</p

Time-dependence in D and S characteristics for pGFPamy and pGFP_Star.

<p>Time-dependence in D and S characteristics for pGFPamy and pGFP_Star.</p

Mean fluorescence and standard deviation of pGFP_Star are reduced to autofluorescence level.

<p>Average mean fluorescence (left) and average standard deviation (right) obtained from the fluorescence distributions of the autofluorescence (AF) of <i>B. subtilis</i> 168 parental strain and cells transformed with pGFPamy or pGFP_Star “empty” vectors at OD_600nm of 5. Results include data of four independent experiments involving two separate clones. Error bars indicate the standard error of the mean.</p

Effect of a single dose of levodopa on sexual response in men and women

From animal research, there is ample evidence for a facilitating effect of dopamine on sexual behavior. In humans, little experimental research has been conducted on the inter-relation between dopamine and sexual response, even less so in women than in men. We investigated the effect of levodopa (100 mg) on sexual response in men and women following a double-blind, placebo-controlled crossover design. Genital and subjective sexual responses were measured as well as somatic motor system activity by means of Achilles tendon (T) reflex modulation. Genital and subjective sexual arousal were not affected by levodopa. However, the drug increased T reflex magnitude in response to sexual stimulation in men, but not in women. These results support the view that dopamine is involved in the energetic aspects of appetitive sexual behavior in men. The observed gender difference in the effect of levodopa is discussed in the perspective of possible dopamine-steroid interactio